Detection of the mcr-1 Colistin Resistance Gene in Carbapenem-Resistant Enterobacteriaceae from Different Hospitals in China

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Polymyxins B and E (also known as colistin) are among some antibiotics of the last resort used to treat serious infections caused by carbapenem-resistant Enterobacteriaceae (CRE). However, the recent discovery of a plasmid-mediated colistin resistance gene, mcr-1, sounded the alarm that last-resort antibiotics may be in jeopardy (1). Of particular concern is the spread of mcr-1 into CRE, creating extensively drug-resistant isolates causing untreatable disease. We have recently reported the cooccurrence of NDM-5 carbapenemase and MCR-1 within the same clinical isolate from a tertiary hospital in eastern China (2). The isolates coproducing MCR-1 and NDM-5 were nonsusceptible to nearly all antimicrobial agents tested (2). It is worrisome that these MCR-1-producing CRE isolates may spread further into hospital settings and within high-risk patients, thereby causing untreatable infections. Here we conducted a molecular screening study for clinical CRE isolates collected from six tertiary hospitals in six provinces in order to explore the dissemination of MCR-1-producing CRE in China.

A total of 264 clinical CRE isolates were collected from six large regional hospitals in northern (Beijing), eastern (Suzhou), southern (Guangzhou), northwestern (Yinchuan), and southwestern (Chengdu and Kunming) China between January 2014 and December 2015. They were isolated from respiratory tract (n = 119), urine (n = 50), blood (n = 38), intra-abdominal (n = 22), skin and soft tissue (n = 17), rectal swab (n = 9), wound (n = 5), and other sites (n = 4) of 251 unique patients and included 160 Klebsiella pneumoniae, 36 Escherichia coli, 19 Enterobacter cloacae, 17 E. aerogenes, 11 K. oxytoca, 5 Citrobacter freundii, 4 Serratia, 3 Morganella morganii, 3 Providencia rettgeri, and 3 K. ozaenae spp. and 3 other species. Species identification was performed using matrix-assisted laser desorption ionization–time of flight mass spectrometry (Bruker Microflex LT). PCR detection of carbapenemase genes (bla_KPC, bla_NDM, bla_VIM, bla_OXA48-like, and bla_IMP) showed that 134 isolates were positive for bla_KPC, 69 for bla_NDM, 18 for bla_IMP, and 7 for bla_VIM, while 59 were negative for any carbapenemase gene. Thirty-one isolates were found to carry more than one carbapenemase. We then tested for the presence of mcr-1 using a previously published PCR method (1) and identified a total of five mcr-1-harboring CRE isolates from four different patients in three different hospitals, including three carbapenem-resistant E. coli isolates and two previously reported sequence type 25 (ST25) K. pneumoniae isolates coproducing MCR-1 and NDM-5 (2, 3). In this report, the clinical and molecular characteristics of the three carbapenem-resistant E. coli isolates are described.

Patient 1 was a male in his late 40s with a medical history of high-level paraplegia and nephropyelitis over 20 years. The patient had had long-term urinary catheterization and had a history of recurring urinary tract infections, for which he was admitted to hospital A in Chengdu, Sichuan (southwestern China), in February 2015. On the first day of admission, an E. coli strain (CDA6) was isolated from a catheter-associated urine specimen. CDA6 was resistant to all β-lactams tested, except for aztreonam, and was resistant to ciprofloxacin, levofloxacin, moxifloxacin, piperacillin-tazobactam, co-trimoxazole, and colistin (Table 1).

Patient 2 was a female who was almost 50 years old who had rectal cancer with liver metastases. The patient underwent rectal cancer surgery and liver tumor resection in early 2015 at a cancer hospital in Beijing. She was admitted to hospital B in Beijing (northern China) a few weeks later due to intra-abdominal infection and liver abscess. On the second day of admission, a multidrug-resistant (defined as resistant to three or more antimicrobial classes) (4) E. coli strain (BJ10) was isolated from the ascites. Susceptibility testing showed that this isolate was resistant to colistin and all β-lactam antimicrobial agents, including imipenem, meropenem, and aztreonam, but remained susceptible to amikacin (Table 1).

Patient 3 was a nearly 50-year-old female with a medical history of cirrhosis lasting more than a decade. In September 2015, the patient was admitted to a local hospital due to symptoms of fatigue, abdominal pain, jaundice, and ascites. One month later, the patient was transferred to hospital B in Beijing. The patient underwent therapeutic plasma exchange 2 weeks later in order to improve liver function. In early November, a multidrug-resistant E. coli isolate (BJ13) was collected from a bile culture. The isolate was resistant or intermediately resistant to all β-lactams, as well as to gentamicin, ciprofloxacin, levofloxacin, moxifloxacin, piperacillin-tazobactam, tetracycline, and colistin (Table 1).

Multilocus sequence typing showed that the three E. coli isolates belonged to three unrelated STs: ST167, ST156, and ST457, respectively (5) (Table 1). PCR and sequencing of the carbapenemase genes revealed that CDA6 and BJ10 carried bla_NDM-5, the same bla_NDM variant we previously reported having found in the two MCR-1-producing ST25 K. pneumoniae isolates (2, 3). BJ13 was negative for all carbapenemase genes tested, but it harbored bla_CTX-M-14 and bla_CMY-2 and had a premature stop codon at amino acid 303 in porin gene ompC, which likely explains its carbapenem resistance.

The mcr-1 gene from isolates CDA6 and BJ13 were successfully transferred to recipient E. coli J53^AZ-R strains by conjugation, whereas conjugation transfer for BJ10 was unsuccessful. Plasmid DNA from BJ10 was therefore extracted and used in electroporation experiments with E. coli DH10B as the recipient, but this transfer was also not successful. To our surprise, results of pulsed-field gel electrophoresis (PFGE) with S1 nuclease treatment (S1-PFGE), followed by Southern hybridization probing with an mcr-1-specific fragment, indicated that the mcr-1 gene in BJ10 is located on the chromosome (data not shown). Whole-genome sequencing of BJ10 was then conducted using an Illumina NextSeq platform to confirm the chromosomal integration of mcr-1. De novo assembly (6) and BLASTn analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed that mcr-1 in BJ10 is located on a Tn9-like composite transposon (tentatively named TnApl), with two directly repeated ISApl1 elements flanking the mcr-1–pap2 gene cassette (Fig. 1). TnApl was integrated in the BJ10 chromosome within the intergenic region between two hypothetical protein genes (corresponding to locus_tag ECSE_1545 and ECSE_1546 in ST156 E. coli strain SE11), with 2-bp (TG) putative target site duplications (Fig. 1). To our best knowledge, this report presents the first description of chromosomal integration of mcr-1 in a composite transposon.

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FIG 1

Chromosomal integration of mcr-1 gene. Arrows denote open reading frames (ORF), with red, gray, yellow, and white arrows denoting mcr-1, pap2, ISApl1, and neighboring chromosomal genes (illustrated by locus_tag in ST156 strain SE11 [GenBank accession no. AP009240]), respectively. The small black arrows adjacent to ISApl1 denote the inverted reverse repeats (IRR). The 2-bp putative target duplicated sequences for TnApl1 are underlined.

The bla_NDM-5 genes from CDA6 and BJ10 were successfully transferred to E. coli J53^AZ-R through conjugation. PCR-based plasmid replicon typing (PBRT) showed that bla_NDM-5 was carried by IncX3 plasmids in both NDM-5-producing isolates (7, 8). The mcr-1-harboring plasmid from CDA6 belonged to the IncX4 group, while the mcr-1 plasmid from BJ13 belonged to IncI2 group (Table 1). S1-PFGE of the transconjugants showed that the mcr-1-harboring plasmids from CDA6 (IncX4) and BJ13 (IncI2) were ∼35 kb and ∼65 kb in size, respectively, while the bla_NDM-5-harboring plasmids from CDA6 and BJ10 were ∼45 kb in size. No ISApl1 gene was found upstream of mcr-1 in CDA6 and BJ13 by PCR, but the downstream pap2 gene was identified in all three isolates. Antimicrobial susceptibility testing showed that the mcr-1 transconjugants were resistant to colistin but susceptible to all other agents tested, suggesting that no additional resistance genes coexist on the mcr-1 plasmids. The bla_NDM-5 transconjugants were resistant to all β-lactams and inhibitors, except for aztreonam (Table 1).

Taking the results together, our current and previous studies have demonstrated the emergence of MCR-1-producing CRE from multiple hospitals in China (2, 3). This study extended our knowledge of emerging mcr-1, and the results show that this resistance gene has spread into different geographic regions in China, has been found in multiple bacterial species, and has moved to the chromosome. Our findings underline the importance of continuous microbiological and molecular surveillance with regard to further dissemination of mcr-1.